Several methods are available for detecting proteins and determining the concentration of a protein in solution. These include dye-binding methods, which are well known in the art, and involve a non-specific reaction in which a protein-complexing dye binds to the protein. The formation of a dye-protein complex causes a change in the optical properties of the dye, such that there is a colour change proportional to the amount of protein present in the sample. Protein-complexing dyes used for in vitro protein quantitation include bromocresol green (Gindler, U.S. Pat. No. 3,884,637), HABA and methyl orange, but these are of limited use as they bind almost exclusively to albumin and generally are not very sensitive.
Other methods to determine protein concentration include the Biuret method (Mokrasch and McGilvery, J. Biol. Chem. (1956). 221, p. 909), in which peptide structures containing at least two peptide linkages are reacted with Cu2+ in alkaline solution to form a violet-coloured chelate complex.
Lowry et al. (J. Lab. Clin. Med. (1951). 39, 663) used a pre-treatment of proteins with an alkaline copper solution, similar to the Biuret method, followed by addition of Folin-Ciocalteu reagent (which contains lithium salts of phosphotungstic and phosphomolybdic acids). The colour produced was a result of the reduction of the phosphotungstic and phosphomolybdic acids to tungsten and molybdenum blue by the Cu-protein complex and by the tryptophan and tyrosine of the protein.
A serious drawback of both the Biuret and Lowry methods is that they cannot tolerate reducing agents that are often present in protein samples.
Dye/protein complex formation using Coomassie Brilliant Blue G-250 as a protein-complexing dye has been described (Bradford U.S. Pat. No. 4,023,933). Coomassie Brilliant Blue dyes will bind to a wide variety of proteins. Moreover, the use of the G-250 dye in the appropriate acid medium results in a protein assay reagent having a sensitivity approximately 100 times greater than the Biuret and conventional dye binding techniques and about 3 to 5 times that of the Lowry method (Bradford U.S. Pat. No. 4,023,933). The use of Coomassie Brilliant Blue G-250 dye in the procedure disclosed in U.S. Pat. No. 4,023,933, the “Bradford Assay”, has many advantages over methods that employ other dyes, including high sensitivity, which permits the use of small sample size and utility when reducing agents are present in a sample.
Coomassie Brilliant Blue G-250 exists in two different colour forms, red and blue. The blue form of the dye is present in neutral and alkaline solution while the red form is present in markedly acid solution (pH 0-1). In acidic solution, Coomassie Brilliant Blue G-250 is present in equilibrium between the red and blue forms; such solutions are brownish in appearance. It is believed that as protein binds to the dye, the dye is brought into a different microenvironment and is then protected from the acid medium that gives the red colour to the dye. The strength of the acid medium is important for protein assay sensitivity using Coomassie dyes, because an increase in the strength of the acid medium causes a significant loss in sensitivity of the assay. The protein-dye complex tends to aggregate, which affects the stability of the colour product. The presence of a solubilising agent, such as ethanol, tends to keep the protein-dye complex from aggregating for a reasonable period of time; however, too much ethanol results in a marked shift to the blue form of the dye, i.e., change of the environment to one which is less polar. It has been postulated that the mechanism of the assay is the binding of a carbanion form of the dye to a less polar environment of the protein. This perhaps also explains the negative effect of large quantities of detergent and of acetone on the assay, since these compounds are generally non-polar in nature and would tend to change the environment of the dye.
The principal drawbacks of the Bradford assay are the effective lack of colour stability for extended periods, largely due to precipitation of the protein-dye complex; the failure to show substantially the same reactivity to different proteins; the failure to follow Beer's law; and, most importantly, the adverse affect on the assay of detergents present in a sample (Bradford, Anal. Biochem., 72 248-254, 1976 and U.S. Pat. No. 4,023,933).
Dye/protein complex formation is also used for staining proteins in gels, such as those used in electrophoresis. For example, the dye Coomassie Brilliant Blue G-250 in perchloric acid solution has been so used (Reisner, A. H. et al. (1975) Anal. Biochem. 64, 509-516).
Currently, several commercial Coomassie-based formulations are available to stain proteins in gels after electrophoretic separation. For many electrophoretic applications, detergents such as SDS are used to facilitate separation of proteins. Because detergents adversely affect the colour change on binding of Coomassie dyes to protein, the detergent must be removed by several wash procedures, resulting in extended and convoluted staining procedures.
Thus a major disadvantage of dye-based protein detection and quantitation, in particular using Lowry assay reagents or Coomassie dyes, is the interference from detergents, surfactants and other amphipathic molecules.
Accordingly, there is a desire for reagents and methods for detection and quantitative determination of protein which have improved tolerance to the presence of detergents in the samples and which have improved protein-dye colour stability.